Atomic Resolution Characterization of Kindlin-2 Binding to Phosphatidylinositol Phosphatases in Lipid Bilayers by Solid-State NMR

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Project Summary: Kindlin-2 (K2) is a peripheral membrane protein which regulates integrin, a key protein that mediates cellular adhesion in humans. The goal of this project is to increase our structural understanding of K2 and the lipid membranes to which it binds, to provide key insights into the regulatory function of K2. The interactions of K2 with cellular membranes are difficult to study because membranes are highly dynamic and lack long-range order. As a result, our ability to study membranes and membrane proteins have been limited despite their potential to be impactful in a wide array of systems. In this proposal we will focus on the signaling lipids phosphatidylinositol phosphates (PIPs) which control the activity of K2. This lipid family is important for human health due to the role of PIPs as regulators of numerous cell processes including nutrient sensing and growth. The misregulation of PIP-dependent pathways is implicated in numerous rare and common diseases, including cancer and diabetes. Mechanistic details of PIP activation and regulation of protein binding partners, like K2, are essential to understanding and combating these diseases. Solid-state NMR is a unique tool for the atomic resolution characterization of the structure and dynamics of lipids and determining the factors that govern lipid-protein interactions. Over the five-year timeline of this proposal I plan to develop NMR experiments for directly investigating K2-lipid interfaces with high sensitivity and specificity. The focus of this study will be the conserved PIP binding domains of K2: a ubiquitin-like F0 domain and a pleckstrin homology (PH) domain. The presence of naturally NMR-active and chemically distinct phosphate groups in the PIPs will serve as a handle to directly probe the binding domain-lipid interface. Characterization of specific interactions between lipid head groups and proteins will be the foundation upon which we examine the structural basis of PIP recognition and the mechanism of K2 activation by PIPs. Beyond studies of the protein-lipid interface, we seek to understand how membrane binding affects the interactions of K2 with partner proteins and itself. Our experiments will take advantage of all the latest improvements in NMR hardware, particularly very-fast magic angle spinning. My group and I are highly experienced in developing novel experiments to assign the chemical shifts and solve structures of protein complexes, membrane proteins, and protein fibrils. This provides us with the tools that are required to tackle the structural aspects of lipid-protein and protein-protein interfaces. We will use an integrative approach in which state-of-the-art computational techniques will guide our experimental work. All-atom molecular dynamics will be used to provide hypotheses that may be tested with NMR and to provide atomic resolution interpretations of experimental data. This work is vital for assisting in the design of therapeutics and diagnostics that target K2 and other PIP binding systems. The results of this project will improve our ability to describe K2-PIP interactions, understand the effects of disease-causing mutations on the regulation of integrins, and propose solutions to some of the largest challenges in human health.